Optimizing diagnostics for galactofuranose containing antigens

Abstract

Disclosed herein are methods of detecting microbial infection in mammalian subjects comprising treatment of a sample and detection of galactofuranose (galF)-containing antigenic components utilizing monoclonal antibodies. The methods disclosed provide for pretreatment of biological samples, such as urine samples, to maximize detection of galF antigens and improvement of sensitivity of galF antigen detection assays. The methods include minimizing intelectin-1 binding to galF antigens and improvement of monoclonal antibody binding. The detection methods are useful for identifying the presence of microbial antigens related to bacterial, fungal, and parasitic pathogens, including Streptococcus pneumoniae, Aspergillus species, Fusarium species, Coccidioides species, Cryptococcus species, Histoplasma species, and Leishmania species.

1 . A method for diagnosing a microbial infection in a mammalian subject suspected of having, having, or susceptible to having a microbial infection by detecting the presence of at least one polysaccharide comprising a galactofuranose residue in a biological sample of the mammalian subject, the method comprising:
 a. providing a biological sample from the subject;
 b. contacting the biological sample with at least one antibody specific for at least one polysaccharide comprising a galactofuranose residue in an effective amount to produce a detectable amount of antibody-polysaccharide complex and 
 c. detecting the presence of at least one antibody-polysaccharide complex, wherein the detection of the presence of at least one antibody-polysaccharide complex is diagnostic of a microbial infection in a mammalian subject. 
   
     
     
         2 . The method of  claim 1 , wherein the microbial infection is selected from the group consisting of a bacterial infection and a fungal infection. 
     
     
         3 . The method of  claim 2 , wherein the bacterial infection is caused by an infection of  Streptococcus pneumoniae . 
     
     
         4 . The method of  claim 2 , wherein the fungal infection comprises an infection caused by an infection of an organism selected from the group consisting of  Aspergillus  species,  Fusarium  species,  Coccidomycoses  species,  Cryptococcus  species, and  Histoplasmosis  species. 
     
     
         5 . The method of  claim 1 , wherein the at least one antibody specific for at least one polysaccharide comprising a galactofuranose residue is selected from the group consisting of monoclonal antibody 205 (MAb 205) comprising a variable heavy (V H ) domain of SEQ ID NO: 1 and a variable light (V L ) domain of SEQ ID NO:2; monoclonal antibody 24 (MAb 24) comprising a V H  domain of SEQ ID NO:3 and a V L  domain of SEQ ID NO:4; monoclonal antibody 686 (MAb 686) comprising a V H  domain of SEQ ID NO:5 and a V L  domain of SEQ ID NO: 6; monoclonal antibody 838 (MAb 838) comprising a V H  domain of SEQ ID NO:7 and a V L  domain of SEQ ID NO:8; and monoclonal antibody 476 (MAb 476) comprising a V H  domain of SEQ ID NO:9 and a V L  domain of SEQ ID NO: 10. 
     
     
         6 . The method of  claim 1 , wherein the biological sample is selected from the group consisting of urine, bronchoalveolar lavage (BAL) fluid, serum, blood, and cerebrospinal fluid (CSF). 
     
     
         7 . The method of  claim 1 , further comprising pre-treating the biological sample to remove an inhibitor that interferes with the detection of the at least one polysaccharide comprising a galactofuranose residue in the biological sample before contacting the biological sample with at least one antibody specific for at least one polysaccharide comprising a galactofuranose residue. 
     
     
         8 . The method of  claim 7 , wherein the pre-treating comprises a step selected from the group consisting of filtering, diluting, and concentrating the biological sample, and combinations thereof. 
     
     
         9 . The method of  claim 8 , wherein the pre-treating includes filtering the biological sample to remove an inhibitor that interferes with the detection of the at least one polysaccharide comprising a galactofuranose residue in the biological sample. 
     
     
         10 . The method of  claim 1 , wherein the diagnosing of a microbial infection in a mammalian subject suspected of having, having, or susceptible to having a microbial infection comprises monitoring a treatment regimen of a microbial infection to determine the efficacy of the treatment regimen. 
     
     
         11 . A lateral flow device for diagnosing a microbial infection in a biological sample of a mammalian subject suspected of having, having, or susceptible to having a microbial infection, the devise comprising:
 (a) a solid layer having a first end, a second end, and a surface on which at least one antibody specific for at least one polysaccharide comprising a galactofuranose residue is deposited in a defined area on the surface of the solid layer;   (b) an agent localized proximal to the first end capable of reacting with the at least one antibody specific for at least one polysaccharide comprising a galactofuranose residue to form a detectable complex;   (c) a flow channel in the solid layer that allows passage of sample liquid from the first end of the solid layer to the second end of the solid layer; and   (d) a second agent localized proximal to the second end capable of reacting with the detectable complex, wherein a detection of the detectable complex diagnoses a microbial infection in the biological sample.   
     
     
         12 . The lateral flow device of  claim 11 , further comprising a case enclosing the solid layer. 
     
     
         13 . The lateral flow device of  claim 12 , further comprising a window in the case enclosing the solid layer to allow sample fluid to contact the solid layer. 
     
     
         14 . The lateral flow device of  claim 12 , further comprising a window in the case allowing viewing of the detection of the detectable complex. 
     
     
         15 . The lateral flow device of  claim 11 , wherein the microbial infection is selected from the group consisting of a bacterial infection and a fungal infection. 
     
     
         16 . The lateral flow device of  claim 15 , wherein the bacterial infection is caused by an infection of  Streptococcus pneumoniae.    
     
     
         17 . The lateral flow device of  claim 15 , wherein the fungal infection comprises an infection caused by an infection of an organism selected from the group consisting of  Aspergillus  species,  Fusarium  species,  Coccidomycoses  species,  Cryptococcus  species, and  Histoplasmosis  species. 
     
     
         18 . The lateral flow device of  claim 11 , wherein the at least one antibody specific for at least one polysaccharide comprising a galactofuranose residue is selected from the group consisting of monoclonal antibody 205 (MAb 205) comprising a variable heavy (V H ) domain of SEQ ID NO: 1 and a variable light (V L ) domain of SEQ ID NO:2; monoclonal antibody 24 (MAb 24) comprising a V H  domain of SEQ ID NO:3 and a V L  domain of SEQ ID NO:4; monoclonal antibody 686 (MAb 686) comprising a V H  domain of SEQ ID NO:5 and a V L  domain of SEQ ID NO:6; monoclonal antibody 838 (MAb 838) comprising a V H  domain of SEQ ID NO:7 and a V L  domain of SEQ ID NO:8; and monoclonal antibody 476 (MAb 476) comprising a V H  domain of SEQ ID NO:9 and a V L  domain of SEQ ID NO: 10. 
     
     
         19 . The lateral flow device of  claim 11 , wherein the biological sample is selected from the group consisting of urine, bronchoalveolar lavage (BAL) fluid, serum, blood, and cerebrospinal fluid (CSF). 
     
     
         20 - 22 . (canceled) 
     
     
         23 . An antibody specific for at least one epitope of a polysaccharide secreted by a microbial organism. 
     
     
         24 - 26 . (canceled) 
     
     
         27 . The kit comprising a antibody of  claims 23 . 
     
     
         28 .- 30 . (canceled)